Human optimized Bacillus anthracis protective antigen

ABSTRACT

The invention relates to a humanized nucleic acid construct from  Bacillus anthracis  protective antigen (PA) gene and method of modifying the gene. The humanized gene, and method of producing it, improves the structural fidelity of expressed protein product, when produced in mammalian host cells, to native, bacterially produced protein. The construct is useful in nucleic acid based vaccine formulations against  B. anthracis.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser. No. 12/916,889, filed 1 Nov. 2010, which claims the benefit of U.S. Provisional Application No. 61/260,656, filed 12 Nov. 2009, which are incorporated herein by reference.

BACKGROUND OF INVENTION

1. Field of Invention

The inventive subject matter relates to a codon optimized nucleic acid sequence of Bacillus anthracis protective antigen (PA). The method of codon optimization of the gene is aimed at improving expression in mammals, including humans, as well as enhancing immunogenicity against endogenously produced PA protein. The inventive construct, incorporated into DNA expression systems, can be useful as a component of immunogenic compositions against B. anthracis, such as vaccines.

2. Background Art

B. anthracis, the etiological agent of anthrax, is a spore-forming, gram positive bacterium. Infection can occur through a variety of routes including cutaneous and gastrointestinal, however, inhalational anthrax is the most widely recognized and feared (Baillie, J. Appl Microbiol., 91: 609-613 (2001)). Following inhalation, the majority of the aerosolized spores are immediately phagocytized by alveolar macrophages and transported through the lymphatic channels to hilar and tracheobronchial lymph nodes. This rapidly leads to the multiplication and systemic circulation of vegetative bacilli. It is believed that en route to these regional lymph nodes the spores begin to germinate and multiply within the macrophage.

Advanced stages of infection are predicated on B. anthracis' anti-phagocytic capsule and the secretion of a tripartite exotoxin consisting of a cell binding component, Protective Antigen (PA), which binds to two enzymatically active subunits: Lethal Factor (LF) or Edema Factor (EF) to form lethal toxin (LeTx) and edema toxin (EdTx), respectively. The currently available licensed human vaccine for B. anthracis (BioThrax) is a filtered extract from B. anthracis absorbed to alum and is primarily composed of PA.

SUMMARY OF THE INVENTION

An object of the invention is a humanized, i.e., codon optimized, DNA construct of Bacillus anthracis protective antigen. The modifications enable efficient translation of PA in mammals, including humans.

Another object of the invention is a method of codon optimization utilizing rare host codons in place of rare bacterial codons, rather than those most highly utilized by the host. This enables ribosomal stalling at appropriate places along the gene to ensure intra-molecular associations occur within the nascent protein similar to that which would occur naturally. Correct folding of PA would result in a more efficacious immune response against naturally occurring B. anthracis expressed PA.

A further object of the invention is a method of human optimization whereby codon optimization does not consist of replacing all bacterial codons throughout the length of the gene with the most highly or frequently used codons in the host cell. Instead, the inventive method utilizes consideration of a number of factors in order to afford increased expression efficiency in a mammalian (e.g., human) host cell, as well as an yielding an expressed protein similar in structure to the native, B. anthracis, PA protein.

The first factor considered is protein expression efficiency. By incorporating codons that are highly utilized in the mammalian host cell for the first 50 codons of the bacterial sequence, the mammalian ribosome will effectively engage the mRNA, decreasing the likelihood of early termination of the ribosome is minimized. Another factor is to maximize the opportunity for correct protein folding. This is afforded by first searching for regions in the native PA sequence where rare codons are utilized in the bacterial gene. Regions were rare codons are heavily utilized may result, in normal, native PA expression, specifically proper folding of the expressed protein. In the modified sequence, these use of rare codons is maintained by substituting rare codons from the human bias table. This would permit the ribosome to stall, where it normally would when expressing native PA in the bacteria, and permit normal folding to occur. Another factor is to ensure against unwarranted deletions of mRNA. Therefore, the bacterial sequence is analyzed to search for ribosomal splice sites to ensure that post-transcriptional machinery of mammalian cells did not delete sections of the mRNA. Finally, an analysis of the bacterial sequence is undertaken to identify any regions of complementarity. These regions are important since they could potentially result in the single stranded RNA folding back on itself resulting in unwanted host cell operations, such as ribosomal stalling or premature termination of translation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1(A-C). Alignment of the human optimized sequence (SEQ ID No.1) and the parent (wild-type (wt)) sequence (SEQ ID No. 3). In FIG. 1, the humanized protective antigen (HoPA) (upper sequence) SEQ ID No. 1 is the human optimized gene and PA (lower sequence) is the parent wild-type gene. Asterisks denote nucleotides that align. FIG. 1 A-C shows alignment of by 1-780; 781-1560; and 1561-2208, respectively.

FIG. 2. Evaluation of DNA vaccines. During this 56 day study A/J mice (n=8 per group) injected IM with pDNAVACCultura2™-HoPA encoding the human optimized PA gene with the tissue plasminogen activator (TPA) signal sequence elicited a robust anti-PA IgG response during the 14 days following the second boost. This response gradually contracted over the final 14 days of the study.

FIG. 3. Anti-PA IgG titers in response to homologous prime-boost-boost with pDNAVACCultra2™-HoPA. Eight groups of mice (n=10) were immunized IM with pDNAVACCultra2™-HoPA on three separate occasions 28 days apart. Control groups were injected with pDNAVACCultra2 without HoPA, the lipid adjuvant dioleoyl phosphatidylethanolamine-dimethyl dioactadecylammoniium bromide (DDAB-DOPE) only, and 10 μg of rPA injected with Alum adjuvant. Titers were low in comparison to the control rPA treated group.

FIG. 4. Anti-PA IgG titers in response to homologous prime-boost with pDNAVACCultra2™-HoPA. Eight groups of mice (n=10) were immunized IM with pDNAVACCultra2™-HoPA on two separate occasions 28 days apart. Control groups were injected with pDNAVACCultra2 without HoPA, the lipid adjuvant (DDAB-DOPE) only, and 10 μg of rPA injected with Alum.

FIG. 5. Efficacy of a homologous prime-boost-boost and a prime-boost with pDNAVACCultra2™-HoPA. Eight groups of mice (n=10) were immunized IM with pDNAVACCultra2™-HoPA two separate occasions 28 days apart. Fourteen days after the last immunization all mice were challenged with LD₅₀s of B. anthracis Sterne strain spores. Survival was significantly improved at 90% with three 100 μg doses of pDNAVACCultra2-HoPA, designated in the figure legend as 7162-HoPA, relative to the lower less frequent doses. Recombinant PA protected 100% of the mice.

FIG. 6. Anti-PA IgG titers and survival in response to homologous prime-boost-boost with humanized protective antigen.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following terms are defined:

An immunogenic composition is a composition, containing one or more antigens, including proteins or peptides or nucleic acid expression systems that express immunogenic proteins or peptides in vivo for the induction of a humoral or cell mediated immune response; a vaccine is an immunogenic composition used to induce protective immunity; a DNA expression system is a molecular system containing plasmid or closed loop DNA containing elements for expressing an inserted DNA sequence as polypeptide; a viral expression system is any viral based system, including viral like particles or viral replicons, containing elements for expressing an inserted DNA sequence as a polypeptide.

Immunization of susceptible individuals through the process of vaccination has long been the most desirable approach to disease prevention. This is particularly important in anthrax since manifestation of the disease results in high mortality. Therefore, it is preferred to prophylactically protect against infection rather than attempt to administer antibiotic post-infection.

The current licensed vaccine in the U.S. for anthrax is a cellular filtrate of B. anthracis that mostly contains PA (Baillie, L., J Appl Microbiol, 91: 609-613 (2001)). Unfortunately, due to the nature of the vaccine, batch to batch variability occurs, resulting in inconsistency in efficacy.

In order to alleviate these problems, recombinant technology has been employed. Nucleic acid based, or DNA vaccines represent a relatively recent and attractive vaccination modality. This interest has been stemmed by their inexpensive and easy production, high stability, and flexibility regarding cloning and delivery method. The basic structure of a DNA vaccine is a plasmid or closed loop of DNA (the plasmid “backbone”) that contains a selectable marker, a mammalian promoter such as the CMV promoter for tissue specific expression, and the gene encoding the antigen of interest, in this case protective antigen (PA).

In principal, the DNA is delivered to either immunologically relevant cells of the skin or to cells that have highly active transcription and translation machinery such as muscle cells where the PA gene is expressed via the CMV promoter, released and displayed from the cell, and hopefully picked up by a scavenging dendritic cell or macrophage.

Unfortunately, DNA vaccines in primates and humans often do not elicit humoral or antibody based responses (Calarota, et al., Lancet, 351: 1320-1325; Coban, et al., Infect Immun., 72: 584-588 (2004); Epstein, et al., Hum Gene Ther, 13: 1551-1560 (2002); Klinman, et al., Curr Top Microbiol Immunol, 247: 131-142 (2000); Wang, et al., Science, 282: 476-480 (1998)) which are critical to surviving an anthrax infection. Several approaches have been developed to enhance the immunogenicity of DNA vaccines including the use of adjuvants, altering the delivery system, modifying the plasmid backbone by including CpG motifs, or altering the codon bias (Leitner, et al., Vaccine, 18: 765-777 (1999); O'Hagan, et al., Nat Rev Drug Discov, 2: 727-735 (2003)).

The current invention relates to a DNA construct, useful in vaccine formulations, utilizing a novel human codon-optimized PA gene sequence. The current invention, unlike previously described methods of DNA optimization, not only permits efficient expression of recombinant protein, but also enables expression of protein tertiary structure, with fidelity to the native bacterially expressed protein. PA expression from B. anthracis is optimized through consideration of a number of factors that enable efficient expression in a mammalian host, e.g., human. The factors considered in the process also improve the likelihood of greater tertiary structural similarity between the expressed recombinant protein and native, bacterially expressed PA. The result is a greater likelihood of a more efficacious induction of adaptive immunity.

Method for Humanizing DNA Sequence

DNA vaccines rely heavily on the natural processes of transcription and translation by the eukaryotic host cell. Bacterial gene structures are very different from the human host and require specialized transcriptional and translational apparatuses. These differences include a lack of introns (noncoding regions that eukaryotes splice out of message RNA), the presence of operons (multiple genes in one message), and a variety of secondary structures within the mRNA that are foreign in eukaryotes (Strugnell, et al., Immunol Cell Bio, 75: 364-369 (1997)). Additionally, bacterial proteins are not glycosylated by the bacterial system but contain amino acid motifs which are efficiently and inappropriately glycosylated by eukaryotic cells. Bacterial mRNA also lacks appropriate structures and sequences to insure an effective half-life in eukaryotic cells.

A important additional difference between eukaryotic and prokaryotic transcriptional/translational systems is the significant differences in codon usage and the arrangement of nucleotides in bacterial mRNA that give rise to codons that are rare in eukaryotic mRNA (Manoj, et al., Crit Rev Clin Lab Sci, 41: 1-39 (2004)). These differences may be explained by the composition of the tRNA pool that is available to the host for translation or the guanine/cytosine (GC) and adenine/thymidine (AT) percentages of the bacterial gene and their similarity to the eukaryotic host (Saler, Nat Rev Drug Discov, 2: 727-735 2003)). Coincident with these differences is the operation of the ribosome and the complex combinations of RNAs and proteins that comprise the translational machinery.

During translation, the ribosome attaches to the mRNA by a specific recognition operation. As the ribosome proceeds down the mRNA it specific codons are recognized leading to a defined assembly of amino acids to ultimately build the nascent protein. Ribosomes have been shown to complete this protein synthesis in a complex manner moving down the mRNA at a varying rate of progression resulting in a multitude of different structural results. If a ribosome slows in its progression, it disconnects from the RNA resulting in premature termination of translation.

Variations in the rate of ribosomal processivity can result in the creation of important structural features. For example, pausing is thought to allow proteins to create protein folds, allowing complex intra-molecular associations to occur. These associations can give rise to proper protein function but also create important immunogenic motifs, that are not present from the linear sequence.

However, when bacterial sequences are expressed from eukaryotic host systems, variations, away from that seen in the bacteria, can result in significant differences in ribosomal procession, glycosylation and even premature termination of translation. The result, therefore, in developing immunogenic compositions, are proteins that may not mimic native protein immune induction.

In developing a more antigenically efficient PA protein, genetic incompatibilities between bacterial and eukaryotic genomes were mitigated by modifying the bacterial sequence in order to conform to optimal codon usage in eukaryotic hosts.

There are many approaches that can be taken in the effort to produce bacterial gene sequences that are translated in human cells more efficiently. The most common is to synthesize the new gene sequence using only the most highly used codon in the host organism. However, this method does not take into account differences between prokaryotic and eukaryotic transcription and translational machinery or the guanine/cytosine (GC) and adenine/thymidine (AT) content of the bacterial gene.

The approach utilized in the current invention is to modify the bacterial gene in order to permit expression resulting in a greater likelihood of maintaining fidelity to the bacterially expressed native protein. This is termed here as “human optimization.” The aim of this approach is to produce a recombinant protein with a greater likelihood of inducing a more efficacious adaptive immune response.

In the inventive method to modify proteins for efficient expression of antigens in a eukaryotic host a number of factors are taken into account. These are summarized as:

-   -   a. efficiency of translation;     -   b. fidelity of protein folding;     -   c. minimize excision of mRNA regions by recognition by the         post-transcriptional machinery of mammalian host cell;     -   d. avoidance of single-stranded RNA folding due to resultant         mRNA sequence complementarity.

In the inventive method, the early (i.e., first region) of the gene utilizes codons most highly utilized by the mammalian host cell. This consideration, therefore, improves the efficiency of gene expression by minimizing the likelihood of early termination of the ribosome. Although the extent of the sequence that is left unaltered varies from gene to gene, the region is typically up to 100 bp.

An important consideration is the fidelity of the tertiary structure and folding of the protein produced in eukaryotic cells to the native, bacterially expressed protein. It is recognized that important immunogenic epitopes are likely to exist beyond the linear or even secondary peptide structure. Rather, proper protein folding can bring amino acids or even peptide sequences, that are normally considerably downstream of each other, into juxtaposition, creating important immunogenic conformational epitopes.

To improve the likelihood of producing these epitopes in the recombinant protein, a search of the native PA sequence is undertake in order to ascertain and identify regions containing relatively heavy concentrations of rare codons. These regions may represent domains with specific folding motifs within the normal, native PA protein. Therefore, retention of these regions in the modified sequence is incumbent upon substituting the rare bacterial codons with complimentary rare codons from the human bias table. This would permit ribosome progression to slow, where it normally would in the bacteria, and permit normal folding to occur.

In order to ensure against unwarranted deletions of mRNA the bacterial sequence is analyzed with the aim of identifying ribosomal splice sites. Alteration of these regions, therefore, will ensure that post-transcriptional machinery of mammalian cells does not inadvertently delete sections of the mRNA

Finally, an analysis of the bacterial sequence is undertaken to identify any regions of complementarity. These regions are of importance since these regions could potentially result in single stranded RNA folding back on itself, resulting in unwanted host cell operations, such as ribosomal stalling or premature termination of translation.

Collectively, the inventive method avoids “over optimization” of the bacterial gene sequence. Instead, the method provides a more deliberate procedure leading to an expressed protein with greater antigenic similarity to native, bacterially expressed protein.

Example Design of Humanized Bacillus anthracis Protective Antigen (PA)

In order to illustrate the inventive method, the human optimization of PA (HoPA) was undertaken. As discussed above, the factors that were considered in the development of the humanized gene sequence. The features of HoPA include:

-   -   a. highly used codons for the first 50 codons of the sequence,         thereby effectively engaging the ribosome and reducing premature         termination;     -   b. using rare codons from the human codon bias table in the same         positions where the wildtype PA gene sequence used rare codons         from the Bacillus anthracis codon usage table thus insuring that         where the bacterial ribosome paused during protein synthesis in         the bacteria, the mammalian ribosome did as well;     -   c. ensuring that regions, where there are many rare codons close         together, are maintained but the actual number of rare codons         reduced in order to minimize the likelihood of ribosomal         progression slowing or stalling;     -   d. a search for cryptic ribosomal splice sites was undertaken to         ensure that the post-transcriptional machinery of the mammalian         cells did not delete sections of the mRNA;     -   e. secondary structure determinations to ensure that the         resulting mRNA did not have long regions of complementarity that         would result in a single stranded RNA that was folded back onto         itself, into a secondary structure that could not be resolved by         the ribosome also leading to premature termination of the         translation process.

Human optimization was performed on the non-proprietary wild-type PA gene (GenBank Accession no. AAA22637.1). In designing the new sequence, the factors, above, were considered and incorporated into the new human optimized sequence (HoPA). The new HoPA gene sequence is illustrated in FIG. 1 (A-C) adjacent to the native sequence. The optimized sequence is also listed in SEQ ID No. 1.

Unlike in the native sequence, the new nucleotide sequence lacks many of the rare codons and motifs that hinder expression in eukaryotes while using human rare codons to emulate the overall spacing of rare codons. An important consideration is to avoid over optimization of the gene sequence. Over optimization may result in the most common eukaryotic codons depleting the available reservoir of normally abundant tRNAs. This process may artificially accelerate the processivity of the ribosome, increasing the chance that the nascent protein will not fold into the proper secondary structure.

The newly synthesized PA gene also included Sap1 restriction sites at the N- and C-terminal ends to allow effective cloning into the multi-cloning site of the pDNAVACCultra2™ (Nature Technology, Lincoln, Nebr.) construct. At the same time, the amino terminal Bacillus leader peptide was eliminated since cloning into pDNAVACCultra2™ places the human TPA leader peptide upstream and in-frame of the PA sequence. This modification effectively increases extracellular trafficking of the recombinant PA (rPA) protein by eukaryotic cells. Ultimately, due to codon redundancy, when both genes are translated they result in the same wild-type amino acid sequence, as illustrated in SEQ ID No. 1.

In order to evaluate the expression of HoPA in a eukaryotic cell line Chinese hamster ovary (CHO) cells strain K1 was transfected with pDNAVACCultra2™-HoPA and pDNAVACCultra2™-HoPA carrying the green fluorescent protein (GFP). Efficient transfection and the transcription/translation of HoPA were verified by the expression of GFP from the modified HoPA-GFP vector. Western blot analysis of supernatants from the transfected CHO-K1 cells after 20 hr demonstrated the presence of PA. This study confirmed that HoPA with it's codon optimizations, could be expressed from a eukaryotic cell line using the host cell machinery.

Referring to FIG. 2, analysis of the mouse antibody response following immunizations with HoPA cloned into NTC's pDNAVACCultra2™ DNA vaccine expression vector demonstrated that the animals had mounted PA specific IgG responses. The efficacy of the human TPA sequence at the beginning of HoPA, to direct the synthesized PA protein out of the cell for detection and processing by circulating immune cells, was evaluated.

In the first study 50 μg pDNAVACCultra2™-HoPA or pDNAVACCultra2™-null (no insert) were mixed with a lipid adjuvant prior to being intramuscularly injected into mice (n=8) on three separate occasions 28 days apart. Serum anti-PA IgG titers were tracked for 56 days and demonstrated that following the second homologous boost on day 28 the rPA specific IgG titer increased from baseline to 33.8 μg/ml (FIG. 2).

In another animal study the efficacy following multiple administrations was evaluated (FIG. 3). In this study efficacy was evaluated after injecting various doses (100, 75, 50, 25, and 12.5 μg) of pDNAVACCultra2™-HoPA once, twice, and three times. Serum IgG titers were tracked as before. Dose and frequency related responses were observed, with the highest antigen specific titers (FIG. 3) achieved with the highest dose of DNA injected three times. The same doses of vaccine given twice (days 28 and 42) (FIG. 4) or once did not generate as robust an IgG response as the triple vaccination schedule or rPA.

Two weeks after the last vaccination the mice were challenged intraperitoneally with 40 LD₅₀s of B. anthracis Sterne strain spores (4.03×10⁵ CFU/mouse). Survival was tracked over the course of 14 days (FIG. 5). Survival was significantly improved (p<0.05) with 100 μg of pDNAVACCultra2™-HoPA (designated as 7162-HoPA in the figure below) when administered three times. Doses less than 100 μg or immunization schedules that lacked a second booster are not efficacious. In comparison, 10 μg of rPA protected 100% of the challenged mice. These results speak to the efficacy of HoPA when cloned into the pDNAVACCultra construct. In FIG. 5, the negative control is the use of the adjuvant Dioleoyl phosphatidylethanolamine-dimethyl dioctadecylammonium bromide (DDAB-DOPE).

FIG. 6 illustrates the immunoglobulin concentration following immunization with humanized PA. As seen in panel A, a significant humoral response is evident following either recombinant PA protein or HoPA. However, significantly less antibody response is seen following immunization with the DNA expressed HoPA than following immunization with rPA protein. The clear dichotomy of immunoglobulin induction seen in FIG. 5 between HoPA (expressed from an administered DNA vector) and rPA protein administration induced between is likely dependent on the expression efficiency of the DNA expression system.

Additionally, antibody induced by HoPA was capable of efficiently neutralizing lethal toxin, as evidenced by toxin neutralization activity (TNA) assay. These results are summarized in Table 1. The assay was conducted as described by Quinn, C. P., et al., J. Infect. Dis. 190: 1228-1236 (2004). Prior to testing, recombinant PA (rPA) and recombinant LF (rLF) were titrated for toxin potency with J774A.1 cells. The concentrations of rPA and rLF that resulted in more than 99% cell lysis at a fixed cell density of 2×10⁴ cells/well were 45.1 and 36.1 ng/ml, respectively. Serum samples were serially diluted starting at 1:50 out to 1:102400 and were assayed in quadruplicate. The resulting serum neutralization curve (antibody dilution factor versus optical density was analyzed with a four-parameter logistic log fit curve. The primary endpoint calculated from this 4-PL curve is the 50% effective antibody dilution (ED₅₀) that protects 50% of the eukaryotic cells in the assay. This value is reported as the reciprocal of the antibody dilution corresponding to the inflection point (“c” parameter) of the four-parameter logistic log fit of the serum neutralization curve. An ED₅₀ greater than 200 is correlated with survival (Pitt, M. L., et al., Vaccine 19: 4768-4773 (2001). Vaccination with pDNAVACCulture2-HoPA three times elicited antibody responses by day 69 (FIG. 6A) with sufficiently high toxin neutralization capacities greater than 200 ED₅₀.

TABLE 1 Mouse ED₅₀ 1.3 587.8 1.4 409.4 1.7 1144.7 1.8 272.2

A further demonstration of the effectiveness of the humoral response induced by HoPA is illustrated in FIG. 6B. In FIG. 6B, mice were immunized with either rPA protein or plasmid-HoPA expression plasmid (pDNAVACCultra™). In FIG. 6B, the survival of mice following anthrax challenge, that had been immunized with HoPA, was equivalent to that observed for the recombinant protein, despite the much higher levels of immunoglobulin induced. The improved efficacy of the immunoglobulin over that induced from rPA protein likely represents the greater similarity of HoPA to native PA structure. Additionally, HoPA may induce higher affinity anti-PA antibody or higher concentrations of antibody specific to regions on the native PA molecule that are more relevant to immune protection. One explanation is that HoPA may induce helper T-cell response to induce IL4. This may suppress IgG2a and IgG2b responses and increase IgG1.

Although the example shown expressed the novel PA in the pDNAVACCultra™ construct, expression of the optimized PA construct can be inserted and expressed by other suitable expression systems. This could include other DNA and viral expression systems. 

What is claimed is:
 1. A method of modifying a recombinant bacterial gene for expression in a mammalian host comprising: a. replacing highly utilized host codons in the first 2 to 5 percent of the total bacterial gene sequence; b. replacing regions of the sequence where there are stretches of three or more rare bacterial codons with rare mammalian host codons.
 2. The method of claim 1, wherein said mammalian host is human.
 3. The method of claim 1, wherein regions of complementarity are identified within said modified bacterial gene sequence that could result in RNA folding back on itself and removing said regions of complementarity by altering the codons while retaining the expressed amino acid at that position in the protein.
 4. The method of claim 1, wherein the bacterial gene is recombinant Bacillus anthracis protective antigen.
 5. The method of claim 1, wherein cryptic ribosomal splice sites are deleted by altering the codons in that region while retaining the expressed amino acid at that position in the protein.
 6. The method of claim 4, wherein the first 50 codons of said recombinant Bacillus anthracis protective antigen are highly utilized human codons. 